The present invention relates generally to the field of genetic engineering and more particularly to genes for receptor tyrosine kinases and their cognate ligands, their insertion into recombinant DNA vectors, and the production of the encoded proteins in recipient strains of microorganisms and recipient eukaryotic cells. More specifically, the present invention is directed to novel ligands, known as TIE ligand-3 and TIE ligand-4, that bind the TIE-2 receptor, as well as to methods of making and using the novel ligands. The invention further provides nucleic acid sequences encoding TIE ligand-3 or TIE ligand-4, methods for the generation of the nucleic acids and the gene products. The novel TIE ligands, as well as nucleic acids encoding them, may be useful in the diagnosis and treatment of certain diseases involving endothelial cells and associated TIE receptors, such as neoplastic diseases involving tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis and inflammatory diseases. In addition, the ligands may be used to promote the proliferation and/or differentiation of hematopoietic stem cells.
Biologically active ligands of the invention may be used to promote the growth, survival, migration, and/or differentiation and/or stabilization or destabilization of cells expressing TIE receptor. Biologically active TIE ligands may be used for the in vitro maintenance of TIE receptor expressing cells in culture. Cells and tissues expressing TIE receptor include, for example, cardiac and vascular endothelial cells, lens epithelium and heart epicardium and early hematopoietic cells. Alternatively, such ligands may be used to support cells which are engineered to express TIE receptor. Further, the ligands and their cognate receptors may be used in assay systems to identify agonists or antagonists of the receptor.
The cellular behavior responsible for the development, maintenance, and repair of differentiated cells and tissues is regulated, in large part, by intercellular signals conveyed via growth factors and similar ligands and their receptors. The receptors are located on the cell surface of responding cells and they bind peptides or polypeptides known as growth factors as well as other hormone-like ligands. The results of this interaction are rapid biochemical changes in the responding cells, as well as a rapid and a long-term readjustment of cellular gene expression. Several receptors associated with various cell surfaces may bind specific growth factors.
The phosphorylation of tyrosine residues in proteins by tyrosine kinases is one of the key modes by which signals are transduced across the plasma membrane. Several currently known protein tyrosine kinase genes encode transmembrane receptors for polypeptide growth factors and hormones such as epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), platelet derived growth factors (PDGF-A and -B), and fibroblast growth factors (FGFs). (Heldin et al., Cell Regulation, 1: 555-566 (1990); Ullrich, et al., Cell, 61: 243-54 (1990)). In each instance, these growth factors exert their action by binding to the extracellular portion of their cognate receptors, which leads to activation of the intrinsic tyrosine kinase present on the cytoplasmic portion of the receptor. Growth factor receptors of endothelial cells are of particular interest due to the possible involvement of growth factors in several important physiological and pathological processes, such as vasculogenesis, angiogenesis, atherosclerosis, and inflammatory diseases. (Folkman, et al. Science, 235: 442-447 (1987)). Also, the receptors of several hematopoietic growth factors are tyrosine kinases; these include c-fms, which is the colony stimulating factor 1 receptor, Sherr, et al., Cell, 41: 665-676 (1985), and c-kit, a primitive hematopoietic growth factor receptor reported in Huang, et al., Cell, 63: 225-33 (1990).
The receptor tyrosine kinases have been divided into evolutionary subfamilies based on the characteristic structure of their ectodomains. (Ulirich, et al. Cell, 61: 243-54 (1990)). Such subfamilies include, EGF receptor-like kinase (subclass I) and insulin receptor-like kinase (subclass II), each of which contains repeated homologous cysteine-rich sequences in their extracellular domains. A single cysteine-rich region is also found in the extracellular domains of the eph-like kinases. Hirai, et al., Science, 238: 1717-1720 (1987); Lindberg, et al. Mol. Cell. Biol., 10: 6316-24 (1990); Lhotak, et al., Mol. Cell. Biol. 11: 2496-2502 (1991). PDGF receptors as well as c-fms and c-kit receptor tyrosine kinases may be grouped into subclass 111; while the FGF receptors form subclass IV. Typical for the members of both of these subclasses are extracellular folding units stabilized by intrachain disulfide bonds. These so-called immunoglobulin (Ig)-like folds are found in the proteins of the immunoglobulin superfamily which contains a wide variety of other cell surface receptors having either cell-bound or soluble ligands. Williams, et al., Ann. Rev. Immunol., 6: 381-405 (1988).
Receptor tyrosine kinases differ in their specificity and affinity. In general, receptor tyrosine kinases are glycoproteins which consist of (1) an extracellular domain capable of binding the specific growth factor(s); (2) a transmembrane domain which usually is an alpha-helical portion of the protein; (3) a juxtamembrane domain where the receptor may be regulated by, e.g., protein phosphorylation; (4) a tyrosine kinase domain which is the enzymatic component of the receptor; and (5) a carboxyterminal tail which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.
Processes such as alternative exon splicing and alternative choice of gene promoter or polyadenylation sites have been reported to be capable of producing several distinct polypeptides from the same gene. These polypeptides may or may not contain the various domains listed above.
As a consequence, some extracellular domains may be expressed as separate, secreted proteins and some forms of the receptors may lack the tyrosine kinase domain and contain only the extracellular domain inserted in the plasma membrane via the transmembrane domain plus a short carboxyl terminal tail.
A gene encoding an endothelial cell transmembrane tyrosine kinase, originally identified by RT-PCR as an unknown tyrosine kinase-homologous cDNA fragment from human leukemia cells, was described by Partanen, et al., Proc. Natl. Acad, Sci. USA, 87: 8913-8917 (1990). This gene and its encoded protein are called xe2x80x9cTIExe2x80x9d which is an abbreviation for xe2x80x9ctyrosine kinase with lg and EGF homology domains.xe2x80x9d Partanen, et al. Mol. Cell. Biol. 12: 1698-1707 (1992).
It has been reported that tie mRNA is present in all human fetal and mouse embryonic tissues. Upon inspection, tie message has been localized to the cardiac and vascular endothelial cells. Specifically, tie mRNA has been localized to the endothelia of blood vessels and endocardium of 9.5 to 18.5 day old mouse embryos. Enhanced tie expression was shown during neovascularization associated with developing ovarian follicles and granulation tissue in skin wounds. Korhonen, et al. Blood 80: 2548-2555 (1992). Thus the TIEs have been suggested to play a role in angiogenesis, which is important for developing treatments for solid tumors and several other angiogenesis-dependent diseases such as diabetic retinopathy, psoriasis, atherosclerosis and arthritis.
Two structurally related rat TIE receptor proteins have been reported to be encoded by distinct genes with related profiles of expression. One gene, termed tie-1, is the rat homolog of human tie. Maisonpierre, et al., Oncogene 8: 1631-1637 (1993). The other gene, tie-2, may be the rat homolog of the murine tek gene which, like tie, has been reported to be expressed in the mouse exclusively in endothelial cells and their presumptive progenitors. Dumont, et al. Oncogene 8: 1293-1301 (1993). The human homolog of lie-2 is described in Ziegler, U.S. Pat. No. 5,447,860 which issued on Sept. 5, 1995 (wherein it is referred to as xe2x80x9corkxe2x80x9d), which is incorporated in its entirety herein.
Both genes were found to be widely expressed in endothelial cells of embryonic and postnatal tissues. Significant levels of tie-2 transcripts were also present in other embryonic cell populations, including lens epithelium, heart epicardium and regions of mesenchyme. Maisonpierre, et al., Oncogene 8: 1631-1637 (1993).
The predominant expression of the TIE receptor in vascular endothelia suggests that TIE plays a role in the development and maintenance of the vascular system. This could include roles in endothelial cell determination, proliferation, differentiation and cell migration and patterning into vascular elements. Analyses of mouse embryos deficient in TIE-2 illustrate its importance in angiogenesis, particularly for vascular network formation in endothelial cells. Sato, T. N., et al., Nature 376:70-74 (1995). In the mature vascular system, the TIEs could function in endothelial cell survival, maintenance and response to pathogenic influences.
The TIE receptors are also expressed in primitive hematopoietic stem cells, B cells and a subset of megakaryocytic cells, thus suggesting the role of ligands which bind these receptors in early hematopoiesis, in the differentiation and/or proliferation of -B cells, and in the megakaryocytic differentiation pathway. Iwama, et al. Biochem. Biophys. Research Communications 195:301-309 (1993); Hashiyama, et al. Blood 87: 93-101 (1996), Batard, et al. Blood 87: 2212-2220 (1996).
Applicants previously identified an angiogenic factor, which was originally called TIE-2 ligand-1 (TL1) but is also referred to as angiopoietin-1 (Ang1), that signals through the TIE-2 receptor and is essential for normal vascular development in the mouse. By homology screening applicants have also identified an Ang1 relative, termed TIE-2 ligand-2 (TL2) or angiopoietin-2 (Ang2), that is a naturally occurring antagonist for Ang1 and the TIE2 receptor. For a description of the cloning and sequencing of TL1 (Ang1) and TL2 (Ang2) as well as for methods of making and uses thereof, reference is hereby made to PCT International Publication No. WO 96/11269 published Apr. 18, 1996 and PCT International Publication No. WO 96/31598 published Oct. 10, 1996 both in the name of Regeneron Pharmaceuticals, Inc.; and S. Davis, et al., Cell 87: 1161-1169 (1996) each of which is hereby incorporated by reference. The absence of Ang1 causes severe vascular abnormalities in the developing mouse embryo. C. Suri, et al., Cell 87: 1171-1180 (1996). Ang1 and Ang2 provide for naturally occurring positive and negative regulators of angiogenesis. Positive or negative regulation of TIE2 is likely to result in different outcomes depending on the combination of simultaneously acting angiogenic signals.
The present invention provides for a composition comprising TIE ligand-3 or TIE ligand-4 substantially free of other proteins. The invention also provides for an isolated nucleic acid molecule encoding TIE ligand-3 and an isolated nucleic acid molecule encoding TIE ligand-4. The isolated nucleic acid may be DNA, cDNA or RNA. The invention also provides for a vector comprising an isolated nucleic acid molecule encoding TIE ligand-3 or TIE ligand-4. The invention further provides for a host-vector system for the production in a suitable host cell of a polypeptide having the biological activity of TIE ligand-3 or TIE ligand-4. The suitable host cell may be bacterial, yeast, insect or mammalian. The invention also provides for a method of producing a polypeptide having the biological activity of TIE ligand-3 or TIE ligand-4 which comprises growing cells of the host-vector system under conditions permitting production of the polypeptide and recovering the polypeptide so produced.
The invention herein described of an isolated nucleic acid molecule encoding TIE ligand-3 or TIE ligand-4 further provides for the development of the ligand, a fragment or derivative thereof, or another molecule which is a receptor agonist or antagonist, as a therapeutic for the treatment of patients suffering from disorders involving cells, tissues or organs which express the TIE receptor. The present invention also provides for an antibody which specifically binds such a therapeutic molecule. The antibody may be monoclonal or polyclonal. The invention also provides for a method of using such a monoclonal or polyclonal antibody to measure the amount of the therapeutic molecule in a sample taken from a patient for purposes of monitoring the course of therapy.
The present invention also provides for an antibody which specifically binds TIE ligand-3 or TIE ligand-4. The antibody may be monoclonal or polyclonal. Thus the invention further provides for therapeutic compositions comprising an antibody which specifically binds TIE ligand-3 or TIE ligand-4 and a pharmaceutically acceptable vehicle. The invention also provides for a method of blocking blood vessel growth in a mammal by administering an effective amount of a therapeutic composition comprising an antibody which specifically binds TIE ligand-3 or TIE ligand-4 in a pharmaceutically acceptable vehicle.
The invention further provides for therapeutic compositions comprising TIE ligand-3 or TIE ligand-4 in a pharmaceutically acceptable vehicle. The invention also provides for a method of promoting neovascularization in a patient by administering an effective amount of a therapeutic composition comprising TIE ligand-3 or TIE ligand-4 in a pharmaceutically acceptable vehicle. In one embodiment, the method may be used to promote wound healing. In another embodiment, the method may be used to treat ischemia. In yet another embodiment, TIE ligand-3 or TIE ligand-4 is used, alone or in combination with other hematopoietic factors, to promote the proliferation or differentiation of hematopoietic stem cells, B cells or megakaryocytic cells.
Alternatively, the invention provides that TIE ligand-3 or TIE ligand-4 may be conjugated to a cytotoxic agent and a therapeutic composition prepared therefrom. The invention further provides for a receptorbody which specifically binds TIE ligand-3 or TIE ligand-4. The invention further provides for therapeutic compositions comprising a receptorbody which specifically binds TIE ligand-3 or TIE ligand-4 in a pharmaceutically acceptable vehicle. The invention also provides for a method of blocking blood vessel growth in a mammal by administering an effective amount of a therapeutic composition comprising a receptorbody which specifically binds TIE ligand-3 or TIE ligand-4 in a pharmaceutically acceptable vehicle.
The invention also provides for a TIE receptor antagonist as well as a method of inhibiting TIE ligand-3 or TIE ligand-4 biological activity in a mammal comprising administering to the mammal an effective amount of a TIE antagonist. According to the invention, the antagonist may be the TIE ligand-3 or TIE ligand-4 as described herein, an antibody or other molecule capable of specifically binding TIE ligand-3, TIE ligand-4 or TIE receptor, or a ligandbody comprising the fibrinogen-like domain of TIE ligand-3 or TIE ligand-4.